Whitefly-transmitted geminiviruses have become a major limiting factor in tomato production in Florida, the Caribbean and much of Latin America. This group of viruses is currently expanding in the Western Hemisphere, and the number of characterized geminiviruses which infect tomato in this region has increased from three to more than 17 over the last 10 years (Polston and Anderson, 1997). This expansion is continuing and reports of new epidemics are appearing almost monthly. Whitefly-transmitted viruses appear alone and in mixed infections with other geminiviruses and other viruses. Whitefly-transmitted geminiviruses are reducing tomato yields in many countries, and total crop losses are not uncommon (Polston and Anderson, 1997). Tomato production in Florida has suffered significant losses (estimated at $125 million in 1990-91) due to tomato mottle virus (ToMoV) infection, which first appeared in 1989. There are no estimates of losses in Puerto Rico due to the tomato geminiviruses, potato yellow mosaic virus (PYMV) and ToMoV, but yields have been reduced significantly (Brown et al., 1995). Tomato yellow leaf curl virus (TYLCV-Is) which caused extensive losses to tomato production in the Dominican Republic (reviewed by Polston and Anderson, 1997) has now been found in Florida (Polston et al., 1999). Incidences of TYLCV-Is are increasing and economic losses were felt this past fall (1998). TYLCV-Is is widespread in Florida, is likely to increase over the next few years and will become a major constraint to tomato production in Florida.
Geminiviruses are very difficult to economically manage in fresh market tomatoes, and practically impossible to manage in processing tomatoes. At this time geminiviruses are managed primarily through the use of a single insecticide, imidacloprid, to reduce the population of the whitefly vector. Tolerance to this insecticide has already been reported from other countries (Cahill et al., 1996; Williams et al., 1996). It may be only a matter of time before imidacloprid loses efficacy in the United States and other locations. The average Florida tomato grower spent approximately $250/acre for insecticides to control ToMoV in 1994 through 1997. These costs are expected to increase significantly as growers' struggle to manage TYLCV-Is. In Caribbean countries geminiviruses have caused many small and medium size tomato growers to go out of business due the increases in costs of production and crop losses. In Israel, where imidacloprid resistance is present, TYLCV-Is is managed by pesticide use plus exclusion; tomatoes are produced in greenhouses enclosed in whitefly-proof screening material or in screened tunnels in the field. The use of these methods are expensive and are often not an economically or horticulturally realistic alternative. The least expensive and most practical control of whitefly-transmitted geminiviruses is the use of resistant cultivars. At this time there are no commercially available resistant tomato cultivars for the geminiviruses native to the Western Hemisphere. There are several cultivars available which have tolerance to TYLCV-Is, however the fruit size and the horticultural attributes of these cultivars are unsuitable for production in Florida.
There are no commercially available ToMoV-resistant tomato cultivars. ToMoV-resistance from Lycopersicon species has been incorporated into tomato (L. esculentum) backgrounds but resistance is closely linked with small fruit size. This linkage has significantly delayed development of resistant plants. Resistance to ToMoV in both tobacco and tomato has been described using mutated coat protein and movement protein genes from ToMoV (Abouzid et al., 1996; Duan et al., 1997a; Duan et al., 1997b; Polston et al., 1996; Sinisterra et al., 1997; Sinisterra et al., 1999). A mutated BC1 gene has been shown to give broad-spectrum resistance (Duan et al., 1997a).
There are few reports suggesting that the gene encoding the geminivirus replication associated protein (Rep) might be used for resistance. There has been a report that a modified ToMoV Rep mutated in a NTP-binding motif was transformed into tomato plants and demonstrated to interfere with viral replication (Stout et al., 1997). Hanson et al. (1995) analyzed phenotypes of BGMV (bean golden mosaic virus) with mutations in a NTP-binding motif of the Rep gene, and demonstrated that the NTP-binding domain is required for replication. They proposed that mutations in this motif may serve in a trans-dominant negative interference scheme for pathogen-derived resistance (also known as “dominant negative mutations”). Resistance to African cassava mosaic geminivirus (ACMV) in Nicotiana benthamiana plants was developed by transformation with ACMV Rep (Hong and Stanley, 1996).
Resistance has been reported with the Rep gene of a monopartite virus, tomato yellow leaf curl virus (TYLCV), a geminivirus only distantly related to ToMoV. Noris et al. (1996) found TYLCV-resistance in N. benthamiana plants using the TYLCV Cl gene with a truncated C-terminal (210 amino acids). However, resistance was overcome with time. Brunetti et al. (1997) transformed tomatoes with the same construct and found that high accumulation of the truncated Rep protein was required for resistance, that high accumulation resulted in a “curled” phenotype, and that the resistance did not extend to an unrelated geminivirus. The plants transformed according to the methods of the subject invention have a normal phenotype and are high yielding as well.